THAT UNWANTED COSMETIC INGREDIENT--BACTERIA • 137 broken the odor had changed and it was anything but the nice homo- geneous product he expected to see. Eventually, a sample was submitted for bacteriological inspection. It was loaded with a relatively pure strain of Pseudomonas aeruginosa, ap- proximately 12,000,000 per milliliter. The president's blood turned to ice and the chemist was about to expire. Further samples of the same batch were similar in appearance and bacterial content as the original. As is often the case, the immediate problem was to correct the situation and keep the production line going. The laboratory at the plant prepared numerous samples containing various preservatives and mixtures of same which were to be tested against this particular strain of Pseudomonas aeru- ginosa. Fortunately, it only required two to three days to give the answer as to which sample was adequately preserved against this organism. Just what the preservative was, I do not know. However, I do know that it required a mixture of preservatives to produce the desired results. Most likely, a synergistic action resulted. An investigation as to where this organism originated was never undertaken, but I can assure you that the kettles, pipe lines, valves and receptacles were all cleaned thoroughly with hot water containing 0.25 per cent formalin. This sounds like a relatively simple problem but, when it strikes a plant in production, one has to work quickly--all the time frantically wondering when the problem wild be solved and whether or not other products being manufactured in the same plant will also become contaminated. Every one--from management down to the laborer--is greatly concerned and, during this time, has very little sleep or mental ease. CASE No. 4 This case differs from the aforementioned ones in that it concerns itself with the synthesis of the active ingredient and, also, the final shampoo for- mulation. It was understood that the active ingredient was made by reacting a polyhydric alcohol with coconut oil fatty acids, sulfating, and then making the salt. A pilot plant batch was prepared and the concentrated, purified, material was stored in a tank for a few days. Upon inspection, it was noticed that the liquid active ingredient had separated into two layers--on the surface was a layer of fatty acids be- neath which was the polyhydric alcohol-water layer. The presence of a slimy appearance, coupled with other characteristics, prompted a bacterio- logical examination. Large quantities of Pseudomonas aeruginosa were found and subsequent experiments showed that this organism could hydrolize the polyhydric alcohol-fatty acid sulfate compound with relative ease. Numerous germicides, antiseptics, and preservatives were investigated

138 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS which finally resulted in a well preserved active ingredient and shampoo-- so it was thought! In fact, the final product could be classified as being sterile. These well preserved shampoos became cloudy within a few days and again separated into two layers. Further bacteriological tests showed that they were free from bacteria--what, then, was going on ? The only logical answer was that there must be an enzyme causing hydrolysis. To prove this, a culture was made of the isolated Pseudomonas aeru- ginosa, treated with formaldehyde to kill the organism, aseptically filtered and stored in the refrigerator. The enzyme formed by the Pseudomonas aeruginosa, called pyocyanase, was in this sterile filtered solution. Simul- taneously, samples of freshly prepared shampoo were sterilized in the auto- clave. Upon cooling, aliquots were then inoculated with the sterile enzyme solution. Those stored at incubation temperature hydrolized overnight while those at room temperature required several days to produce hy- drolysis. With this information, it was evident that the appropriate preservative must be incorporated into the concentrated active ingredients at an early stage during manufacture. In this manner, the bacteria are retarded or killed--preferably the latter--and, since the enzyme was not given a chance to be formed, the active ingredient did not hydro]ize. SufFicient preservative was added to the concentrated active ingredient at this stage so that the finished formulated shampoo contained enough preservative to retard any bacterial growth. It required a year of steady work to solve this problem to the point where a stable, marketable shampoo was produced. One can readily see how expensive a problem of this sort can be, not only from the standpoint of solving the problem but, also, from the sales lost during this period. However, this company can consider itself fortunate in that the trouble was encountered before marketing rather than afterwards. C^sE No. 5 A marketed cream shampoo exhibited numerous little black specks on the surface. Every one visually examining the product considered these specks to be incipient mold growth. However, extensive bacteriological work showed that they were caused by the organism zflcaligenes viscosus. Eventually, the source of the contamination was traced to the sodium lauryl sulfate and was isolated only after frantic efforts. Extreme difficulty was encountered in growing the organism until it reached the point where the discolored spots were streaked on every type of agar media in the laboratory--preparing two sets of plates--incubating one at room temperature and the other at 37øC. This technique is known as "the last resort."